Hyperthermia or heat shock therapy is currently being utilized in phase II/III clinical trials, either alone or in combination with radiation or chemotherapy, for the treatment of various cancers. Unfortunately, though intense heat shock induces apoptosis, the underlying mechanisms remain controversial and unclear. We have recently shown that heat shock does not require any of the known initiator caspases or their activating complexes to induce apoptosis. We now hypothesize that heat shock induces cell death, in part by stimulating rapid endo-lysosomal membrane permeabilization (ELMP), which coincides with cytosolic acidification and release of cathepsins into the cytoplasm, both of which participate in the processing of procaspase-3 and the proapoptotic BH3-only protein Bid. Importantly, heat shock-induced mitochondrial outer membrane permeabilization (MOMP) is essential for cell death, as cells that overexpress Bcl-2-or are deficient in Bid or the proapoptotic Bcl-2 family members Bax and Bak-are resistant to cell death. MOMP is critical for cell death most likely because it facilitates the release of inhibitor of apoptosis (IAP) antagonists, which in turn promote caspase activity. Remarkably, heat shock also requires c-Jun N-terminal kinases (JNKs) to induce MOMP, and we find that JNKs are activated through a highly novel pathway involving the formation of so-called cytoplasmic stress granules (SGs). We hypothesize that formation of SGs is initiated through heat-induced aggregation of the RNA binding protein TIA-1, and that SGs in turn activate JNKs through a tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) and TGF2- activating kinase 1 (TAK1)-dependent pathway. We further speculate that JNKs sensitize cells to heat shock-induced MOMP by activating additional BH3-only proteins, such as Bim, and/or by inhibiting specific antiapoptotic Bcl-2 family members, such as Bcl-xL or Mcl-1. In the following three specific aims, we propose (1) to determine if ELMP elicits cytosolic acidification and to determine how ELMP is regulated by heat shock protein 70 (Hsp70), (2) to establish the roles of cathepsins in the activation of procaspase-3 and Bid, and (3) to characterize the unique SG-TRAF2-TAK1-MAPKK-JNK pathway, its regulation by Hsp70, and the downstream targets of JNK following heat shock. In summary, this grant proposal would determine the basic mechanisms of heat shock-induced apoptosis, which are highly relevant given the renewed interest in hyperthermia as a clinically useful treatment option. PUBLIC HEALTH RELEVANCE: The focus of this grant project is to unravel the cellular mechanisms that mediate heat shock-induced cell death. Heat shock, or hyperthermia, is currently being used in clinical trials, either alone or as an adjunct to chemo- or radiotherapy for the treatment of various cancers. Unfortunately, very little is known about how heat shock kills cells. Our preliminary work in this area suggests that heat shock induces cell death through mechanisms that are very different from other types of stimuli. Thus, it is anticipated that the work proposed herein will significantly improve our understanding of how heat shock works in the clinic and may shed light on new pathways that can be exploited therapeutically in the future.